Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander gamma-H2AX induction. In this paper, we used normal (rho(+)) and mtDNA-depleted (rho(0)) human-hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with gamma-H2AX, we found that the fraction of gamma-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated rho(+) cells at 10 min post-irradiation (rho(+) ICCM10min) caused larger increases of bystander gamma-H2AX induction comparing to rho(0) ICCM10min, which only caused a slight increase of bystander gamma-H2AX induction. The rho(+) ICCM10min could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with rho(0) ICCM10min. We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched gamma-H2AX induction by rho(+) ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59(-) gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of rho(+) ICCM-treated bystander cells. In conclusion, the work presented here suggested that upregulation of the mitochondria-de pendent ROS might be very important in mediating genotoxicity of bystander effects. (c) 2009 Elsevier B.V. All rights reserved.